Gluten is a common complex of proteins found in certain grass-related grains, including wheat, barley, and rye. Gluten is a mixture of proteins comprising gliadin and glutenin. A 33-mer peptide derived from α-2 gliadin (residues 57-89), which is not digested in the human digestive system, has been identified as an initiator of the inflammatory response to gluten in, for example, Celiac disease. The 33-mer derived from α-2 gliadin is particularly rich in proline and glutamine residues. It stimulates a T-cell immune response in susceptible subjects, resulting in an inflammation that damages the intestinal wall. This, in turn, impairs the ability of the intestine to absorb nutrients, leading to malnutrition and a variety of other symptoms.
Gluten intolerance, or gluten sensitivity, is a collective term which includes all kinds of sensitivity to gluten. A small proportion of gluten intolerant people will test positive for Celiac disease. The standard diagnostic for celiac disease is villus atrophy detected in duodenal biopsies. In addition, antibodies to tissue transglutaminase (tTG) and gliadin appear in almost 100% of the patients with active Celiac disease, and the presence of such antibodies, particularly of the IgA class, has been used in diagnosis of the disease. The large majority of Celiac patients express the HLA-DQ2 [DQA1*0501, DQB1*02] and/or DQ8 [DQA1*0301, DQB1*0302] molecules. Clinical symptoms of Celiac disease include, for example, fatigue, chronic diarrhea, malabsorption of nutrients, weight loss, abdominal distension, anemia, as well as a substantially enhanced risk for the development of osteoporosis and intestinal malignancies (lymphoma and carcinoma).
However, most gluten intolerant, or gluten sensitive, people test negative (or inconclusive) for Celiac disease. Up to approximately 15% of the population (or 1 in 7 people) are non-Celiac gluten sensitive or non-Celiac gluten intolerant. These subjects are non-Celiac gluten sensitive or non-Celiac gluten intolerant in that they suffer symptoms and illness similar to celiac disease patients without meeting diagnostic criteria for celiac disease. Thus, non-Celiac gluten sensitivity (“NCGS”), non-Celiac gluten intolerance (“NCGI”), or gluten related disease (“GRD”), refer to a condition or disorder in which individuals suffer symptoms very similarly to people with Celiac disease, but the diagnostic tests (including blood tests) which identify and diagnose Celiac disease are negative or inconclusive. Thus, as much as 15% of the US population (or 1 in 7 people) may have NCGS.
Moreover, 15-30 million people in United States buy gluten-free products in an attempt to reduce gluten exposure for a variety of reasons.
U.S. Pat. No. 7,320,788 (“Shan”) has proposed administering certain enzymes, termed “glutenases,” to Celiac or dermatitis herpetiformis patients. According to Shan, glutenases include endoproteases found in wheat, barley, and rye, such as an endoprotease from Hordeum vulgare; prolyl endopeptidases (“PEP”), and specifically PEP from Flavobacterium meningoscepticum (Genbank ID# D10980) and Myxococcus xanthus (Genbank ID# AF127082); and brush border enzymes that catalyze the removal of dipeptides, including dipeptidyl peptidase IV and dipeptidyl carboxypeptidase. Shan also mentions that the X-Pro dipeptidase from Aspergillus oryzae (GenBank ID# BD191984) and the carboxypeptidase from Aspergillus saitoi (GenBank ID# D25288) can improve gluten digestion in the Celiac intestine. Shan teaches that the oligopeptides such as Gly-Pro-pNA, Z-Gly-Pro-pNA, and Hip-His-Leu can be used to determine whether a candidate enzyme will digest a toxic gluten oligopeptide. In fact, Shan teaches that the dose of a PEP enzyme is determined by the amount of that enzyme required to hydrolyze 1 micromol Z-Gly-Pro-pNA.
U.S. Pat. No. 7,534,426 (“Piper”) is directed to methods of determining the therapeutic efficacy of candidate glutenase enzyme by detecting the ability of a candidate enzyme to digest one or more selected oligopeptides. US Patent Application Publication No. 2008/0213245 (“Hausch”) refers to enzyme treatment of foodstuffs and also mentions screening methods that employ toxic oligopeptides to identify active glutenases. Like Shan, both Piper and Hausch mention PEPs for treatment of Celiac Sprue and/or dermatitis herpetiformis and teach that the oligopeptide Z-Gly-Pro-pNA can be used to determine whether a candidate enzyme will digest a toxic gluten oligopeptide. U.S. Pat. No. 7,462,688 (“Khosla”) is directed to methods for diagnosing Celiac disease and/or dermatitis herpetiformis by detecting toxic gluten oligopeptides, or T cells and/or antibodies reactive thereto. Kholsa suggests treating such patients by administering peptides that interfere with the binding of toxic gluten oligopeptides to T cells and/or HLA molecules.
International Publication No. WO 2005/027953 (“Edens”) is directed to processes for the proteolytic hydrolysis of a peptide or a polypeptide and mentions using proline specific endopeptidases, such as PEP from Aspergillus species, to produce food devoid of Celiac related epitopes. Likewise, U.S. Pat. No. 7,563,864 (“Marti”) mentions an in vitro proteolytic protocol to detoxify gluten employing pepsin, trypsin/chymotrypsin, elastase, carboxypeptidase A, PEP, and rat brush border membrane enzymes. Rizello et al. (Applied and Environmental Microbiology, July 2007, p. 4499-4507) uses a combination of lactobacilli and proteases from Aspergillus oryzae (supplied by BIO-CAT) to reduce gluten concentration during food processing. Doumas et al. (Applied and Environmental Microbiology, December 1998, p. 4809-4815) provides the sequence of Aspergillus oryzae prolyl dipeptidyl peptidase (DPPIV) and suggests that the DPPIV enzyme may be of importance in industrial hydrolysis of wheat gluten-based substrates.
Ehren et al. (PLoS ONE 4(7):e6313) discuss a food-grade enzyme preparation with modest gluten detoxification properties. Ehren et al. note that aspergillopepsin from Aspergillus niger markedly enhances gluten digestion. Ehren et al. employed peptidase P from Aspergillus oryzae, which contains the exopeptidase DPPIV, to augment the extent to which ASP hydrolyzes gluten. Ehren et al. teach that DPPIV alone is unable to detoxify gluten oligopeptides, including a synthetic 33-mer from α2-gliadin or the 26-mer from γ5-gliadin and that DPPIV is ineffective at low pH and, therefore, would not be effective in vivo in the absence of an antacid adjuvant.
“Oral Papain in Gluten Intolerance” (“Messer et al.”) mentions that the treatment of a patient regarded as having celiac disease with oral papain resulted in the patient being able to consume a gluten-containing diet with no further symptoms of celiac disease. Australian Patent Application No. 2008100719 (“Cornell and Stelmasiak”) mentions compositions and methods for the prophylaxis or treatment of celiac disease. Cornell et al. mentions compositions and methods that include an extract of papaya resin or a functional analogue thereof “Papaya latex enzymes capable of detoxification of gliadin” (“Cornell et al.”) mentions that the activity of papaya is due largely to caricain, and to a lesser extent chymopapain and glutamine cyclotransferase.